Purification of antibiotics with water soluble salts of water insoluble carboxylic acids



Patented Mar. 10, 1953 UNITED STATS TENT OFFICE PURIFICATION or ANTIBIOTICS WITH WATER SOLUBLE SALTS OF WATER INSOLUBLE CARBOXYLIC ACIDS No Drawing. Application July 19, 1947, Serial No. 762,206

18 Claims. (Cl. 260210) '1 This invention relates to basic antibiotics of the streptomycin-type, i. e., to members of the genus composed of streptomycin and antibioticallyactive basic compounds which (like streptomycin) are capable of forming water-soluble salts with acids such as sulfuric and water-insoluble salts with organic-base-precipitating reagents (e. g., derivatives of streptomycin, such as dihydrostreptomycin and similarly-acting antibiotics, such as streptothricin).

In 1944, Schatz, Bugle, and Waksman (Proc. Soc. Exp. Biol. Med. 1944, 57, 244) showed that a potent antibiotic, termed streptomycin, was formed during the growth of the organism Actinomyces grz'seus (now called Streptomyces griseus); and this antibiotic has since been demonstrated to be of high clinical utility.

It was later found that a number of streptomycins are formed at the same time. The first streptomycin obtained in pure crystalline form (as a reineckate) and fully characterized (Wintersteiner and Fried application, Serial No. 666,541, filed May 1, 1946) is now referred to as streptomycin A; and the second streptomycin characterized (Fried and Titus'application Serial No. 737,400, filed March 26, 1947) is now referred to as streptomycin B. Moreover, there are indications that still other streptomycins are formed at the same time and/or may be formed at the same time by changes in the culture conditions; and it is intended that each of such antibiotics and any mixtures thereof (whether in the form of the free base or water-soluble salt thereof) be comprehended by the term streptomycin when employed unmodified hereinafter in the specification and claims.

Streptomycin has been purified heretofore by various methods, all of which were complex and/or inefilcient (and consequently expensive). For example, a method widely employed prior to this invention essentially comprised the following steps: (1) treating a primary streptomycincontaining liquid with an activated charcoal, which selectively adsorbs the streptomycin; (2) eluting the streptomycin from the charcoal with an aqueous, water-soluble mineral acid, preferably at a slightly elevated temperature (say about 30-50" 0.); (3) treating the eluate with an organic-base precipitating reagent,- preferably phosphotungstic acid; and (4) decomposing the precipitate. [The term primary streptomycincontaining liquid comprehends, inter alia: (a) the culture liquid obtained by growing Streptomyces griseus under conditions and in a medium 7 suitable for the production of streptomycin, and

removing the solids from the medium; (b) the culture liquid of enhanced potency obtained by acidification of such culture (with hydrochloric or sulfuric acid, for example), such liquid being neutralized; and (c) the liquid obtainedby acidextraction of the solids separated from such culture, such liquid being neutralized] f It is the object of this invention to provide simple, efiicient, and otherwise advantageous methods of purifying basic antibioticsof the streptomycin type-especially streptomycin; and

it is a further object of this invention to provide methods of preparing certain salt-type derivatives of basic antibiotics of the streptomycin type.

The methods of this invention essentially comprise interacting a basic antibiotic of the streptomycin-type (especially streptomycin) with a water-soluble salt of a substantially water-insoluble carboxylic acid (preferably with a watersoluble alkali salt-which term is to be understood herein as embracing ammonium saltof a higher fatty acid) in a solvent for the reactants (especially water). The formed salt-type combination of the antibiotic and the carboxylic acid is relatively insoluble in water, and precipitates as formed when water is employed as the solvent for the reactants. When the employed solvent for the reactants is one (such as methanol) in which the salt-type combination is s0luble,.-the formed salt-type combination is recovered by removing the solvent or adding a miscible nonsolvent for the salt-type combination (suchas water). The purification methods of this invention essentially comprise treating an impure basic antibiotic of the streptomycin type with a watersoluble salt of a substantially water-insoluble carboxylic acid in a solvent for the reactants, recovering the formed salt-type combination of the antibiotic and the carboxylic acid, and converting the latter combination into a water-soluble salt of the antibiotic. Such conversion may be effected, for example, by dissolving the salt-type derivative in a substantially water-immiscible organic solvent for the carboxylic 'acid, intimately contacting the organic solvent solution with an aqueous, water-soluble, relatively-strong acid (especially with an aqueous, water soluble, relatively strong mineral acid), and recovering the aqueous phase; the salt recovered from the aqueous solution thus obtained being considerably purer.

The water-soluble salts of substantially waterinsoluble carboxylic acids utilizable in the practice of this invention include the alkali-metal (which term is to be understood herein as'emantibiotic action.

bracing ammonium) salts of the following acids, inter alia: aliphatic acids, such as lauric, 2- ethyl-hexoic, sorbic, caprylic, palmitic, and oleic acidsespecially coconut-oil fatty acids, palmkernel fatty acids, or other technical lauric acid (i. e., mixture of fatty acids having an average molecular weight approximating that of lauric acid); aromatic acids, such as benzoic, phenylacetic, p-amino-benzoic, 3,5-diriitro-benzoi cinnamic, and a decyl -cinnamic; cycloaliphatic acids, such as camphoricand naphthenic; and heterocyclic acids, such as indoleacetic and furoic.

The substantially water-immiscible organic solvents for substantially water-insoluble carboxylic acids utilizable in the practice of this invention include, inter alia: aliphatic alcohols, such as n-amyl alcohol (l-pentanol), n-buta-nol (l-butanol), methyl-isopropyl carbino'l, methylisobutyl carbinol, .isobutyl carbinol, Z-ethyl- 'hexanol, and alcohol mixtures, such as refined i'ermentation-amyla1coholor a commercial mixture of synthetic amyl alcohols (e. g., Pentasol);

aliphatic alcohol esters of lower fatty acids, such as the, acetate of methyl-isobutyl carbinol; aliphatic ketones, such as methyl-isobutyl ketone; aliphatic ethers, such as di-n-butyl ether and diethyl ether; hydrocarbons, such as benzene and toluene; and halogenated hydrocarbons, such as chloroform and ethylene dichloride.

Among the water-soluble, relatively-strong acids'utilizable in the practice of this invention are sulfuric, hydrochloric, tartaric, phosphoric, oxalic, and citric.

In one-embodiment of the invention, a culture filtrate containing a basic antibiotic of the streptomycin type is mixed with an aqueous solution of a water-soluble salt of a substantially waterinsoluble 'carboxylic acid, and the precipitate "antibiotic treated is a culture filtrate, the pre-- cipitate obtained is washed with water before further. treatment, the wash removing a considerable amount of. inactive solids but practically none of the antibiotic.

The'precipitationof the salt-type combination of the antibiotic and the carboxylic acid is dependent on, the concentration of the reactants and the pH of, the reaction medium. Maximum precipitation is obtained when the concentrations of'the antibiotic and/or the water-soluble carboxylic, acid salt in the reaction medium are high and the pH of the reaction mixture is between about 6.0 and about 8.0, and preferably around 7.0.

Alternatively, the salt-type combination of the antibiotic and the carboxylic acid formed may be usedas a therapeutic agent per so. These salt-type derivatives of the antibiotics are, in

general, oil-soluble or oil-dispersible; and they maybe orally administered either per se or in oily media for the treatment of intestinal disorders: (relying on intestinal processes to liberate the: antibiotic in water-soluble form), or parenterally administered in oil media for prolonged Where production of the salta type combination of the antibiotic and the substantially water-insoluble carboxylic acid, rather than purification of the antibiotic, is the objective, one may employ a reconstituted solution of the antibiotic (e. g., an aqueous solution of the highly-purified or pure antibiotic).

The amount of carboxylic acid salt employed should desirably be sufficient to interact with all of the antibiotic in the solution treated, and preferably should be in excess.

'Theprecipitated salt-type combination of the antibiotic and the carboxylic acid may be further treated as follows (whether it is to be used as a therapeutic agent per se or converted into a water-soluble salt): the precipitated salt-type derivative is dissolved in a suitable solvent, e. g., tertiary butanol, and freeze-dried (i. e., frozen, and subjected to a high vacuum to sublime oil the solvent); preferably, the precipitated salt-type derivative is first washed with water, dissolved in tertiary butanol (for example), precipitated from the solution (by addition of acetone, or

other non-solvent for the salt-type derivative which is miscible with the tertiary butanol'), redissolved in tertiary butanol, and freeze-dried.

The following examples are illustrative of the invention (all solutions or dilutions referredto without identification of the solvent or diluent being solutionsin, or dilutions with, water):

Example 1 (a) 15.5 g. streptomycin hydrochloride, having apotency of 528 units/mg, is dissolved in 400' ml. water, and the solution is adjusted to pH 7 by addition of sodium hydroxide solution; to this solution is added a solution'of sodium laurate prepared by digesting 39.52 g. lauric acid with 33.9 ml. 5.8 N sodium hydroxide solution; and the curdy precipitate formed is separated by centrifugation.

(b) The precipitate (a salt-type combination of streptomycin and lauric acid) is washed with water, and "dissolved in tertiary butanol; the salttype derivative is "then 'repreci'pitated by addition of acetone, and redissolved in tertiary butanol, and the solution is freeze-dried, yielding about 8.3 g. of the substantially-pure salttypederivative having a potency of 290 units/mg. Thesalt-type derivative may be used as a therapeutic agent per se, or converted into a watersoluble salt of streptomycin, e'. g., as described in section (b) of the following example.

Eaxcmple 2 300 ml. refined fermentation-amyl alcohol, and

the-solution is washed four times, each time with one-fourth its volume distilled water; the washed amyl alcohol solution is then extracted with onefourth its volume of third-normalsulfuric acid, and then' with two portions of distilled water, each one-fourth the volume of the amyl alcohol solution; the three extracts are combined and washed with one-fourth their combined volume ct freshamy-l alcohol Q'to remove" any entrained lauric acid); and the washed extract is separately treated with three portions of an adsorbent carbon (e.'g., Darco (3-60), 0.5 g. carbon per million units streptomycin in the extract being used for each portion. The extract-is adjusted topI-I 5.5-6.0 by addition ofbarium hydroxide solution; and after standing several hours, the'extract is filtered toremove precipitated barium sulfate, partially concentrated at low temperature in vacuo, and freeze-dried. 1 The streptomycin sulfate thus obtained has a potency of about 400 units/mg.

Example 3 (a) 36.33 g. streptomycin sulfate having a potency of 544 units/mg. is dissolved in 988 ml. water, and the solution is adjusted to pH 7 with aqueous sodium hydroxide solution; to this solution is added a solution of sodium laurate prepared as described in section (a) of Example 1; and the curdy precipitate formed is separated by centrifugation. v

(b) The precipitate (a salt-type combination of streptomycin and lauric acid) is treated as described in section (b) ofExample 1, yielding about 21.0 g. of the substantially-pure salt-type derivative-having a potency of 320 units/mg.

' Example 4 ta) 200ml. of a streptomycin-containing culture filtrate, having a potency of 400 units/ml., is adjusted to pH 7.15 byaddition of sodium hydroxide solution; a concentrated aqueous solution of sodium laurate (prepared by warming equivalent quantities of sodium hydroxide solu- A tion and lauric acid) .is added, in the proportion of 10 g. lauric acid per g. streptomycin base; and the mixture is agitated for a half hour and then centrifuged. The semi-solid product obtained (a salt-type combination of streptomycin.

and lauric acid) contains about 60.5% of the activity of the culture filtrate.

Sodium laurate in the proportions of 5 g. and

g. lauric acid per g. streptomycin base precipitates respectively about 12.2% and 33.4% of the culture filtrate activity.

(b) The salt-type derivative of streptomycin may be purified as described in section (b) of Example 1 and/or used as a therapeutic'per se,

or converted into a water-soluble salt of streptomycin, e. g., as described in section (b) of Example 2.

Eirample 5 (a) 1 g. dihydrostreptomycin sulfate, having a potency of 531 units/mg, isdissolved in ml. water, and the solution is adjusted to pH 7 by addition of 10% sodium hydroxide solution; to this solution is added 25 ml. of sodium laurate solution, prepared by treating 39.5 g. coconutoil fatty acids with 34 ml. of 20% sodium hydroxide solution and diluting to 200 mluwith water; and the precipitate formed is separated by centrifugation. 1

(b) The precipitate (a salt-type combination of dihydrostreptomycin and coconut-oil fatty acids) is washed with water, and dissolved in tertiary butanol; the salt-type derivative is then reprecipitated by addition of acetone, and the precipitate is redissolved in tertiary butariol; and the solution is freeze-dried, yielding about 160 mg. of the substantially-pure salt-type derivative having a potency of about 74 units/mg. and a nitrogen content of about 5.41%. It may be used as a therapeutic agent per se, or converted into a water-soluble salt of dihydro-streptomycin, e. g'., as described in section (b) of Example 2. I

The supernate remaining on separation of the acetone-formed precipitate gives a further precipitate on standing in the cold, which is separated and dissolved in tertiary butanol; and the solution is freeze-dried, yielding about 380 mg. additional salt-type derivative having a potency of about 55 units/mgqand a'nitrogen content of about 2.31%. 7 I

The lauric acid and coconut-oil fatty acids employed in the foregoing examples may be replaced by any other of the hereinbefore-mentioned substantially water-insoluble carboxylic acids; the refined fermentation-amyl-alcohol used in Example 2 may be replaced by any other of the hereinbefore-mentioned substantially water-imacid), or a streptothricin hydrochloride solution,

inter alia. Also, one can treat a solution of one of the following pure orsubstantially-pure basic antibiotics of the streptomycin type: streptomycin A, streptomycinB, dihydrostreptomycin A, and dihydrostreptomycin B.

The basic antibiotic of the streptomycin type purified by the method of this inventionmay be further purified by repetition of the purification method of this invention. Also, it may be preliminarily purified or further purified by any other method, especiallyby one of the following: (I) intimately contacting an aqueous solution of the antibiotic with a substantially waterinsoluble carboxylic acid and a substantially water-immiscible organic solvent for the carboxylic acid, recovering the organic-solvent phase, intimately contacting the organic-solvent solution with an aqueous, water-soluble, relabasic-inorganic-acid type and a substantially water-immiscible organic solvent'ior soaps, recovering the organic solvent phase, and converting the salt-type derivative of the antibiotic therein into a Water-soluble salt of the antibiotic (cf. application Serial No. 767,851, filed August 9, 1947, now Patent No. 2,537,933, dated January-9, 1951).

The invention may be variously otherwise embodied within the scope of the appended claims.

We claim:

1. The method which comprises interacting an antibiotic of the group consisting of streptomycin, dihydrcstreptomycin, streptothricin, and

water-soluble salts of these with a water-soluble salt of a substantially water-insoluble carboxylic acid in a solvent for the reactants.

The method which comprises interacting an antibiotic of the group consisting of streptomycin, dihydrostreptomycin, streptothricin, and water-soluble salts of these with a water-soluble saltof a substantially water-insoluble carboxylic acid in an aqueous medium.

3. The method which comprises interacting a water-soluble salt of streptomycin with a watersoluble salt of a substantially water-insoluble carboxylic acid in an aqueous medium.-

4. The method of purifying an antibiotic oi" the-group consisting of streptomycin, dihydrostreptomycin, streptothricin, and water-soluble salts of these which comprises treating an impure antibiotic of that group with a water-soluble salt of a substantially water-insoluble carboxylic acid in a solvent for th reactants, recovering the formed salt-type. combination of the antibiotic and the carboxylic acid, and converting the latter combination into a water-soluble salt of the antibiotic.

5. The method of purifying an antibiotic of the group consisting of streptomycin, dihydrostreptomycin, streptothricin, and water-soluble salts of these which comprises treating an aqueous so lution of an impure antibiotic of that group with a water solublesalt of a substantially water-insoluble carboxylic acid, recovering the precipitated salt-type combination of the antibiotic and the carboxylic acid, and convertingthe latter combination into a water-soluble salt of the antibiotic.

6. The method of purifying streptomycin, which comprises treating an aqueous solution of an impure water-soluble salt of streptomycinwith a water-soluble salt of a substantially water-insoluble carboxylic acid, recovering the precipitated salt-type combination of streptomycin and the carboxylic acid, and converting the latter combination into a water-soluble salt of streptomycin.

7. The method which comprises interacting a water-soluble salt of streptomycin with a. watersoluble alkali salt of a higher fatty acid in an aqueous medium.

8; The method which comprises interacting a water-soluble: salt of streptomycin with sodium laurate in an aqueous medium.

9. The method of purifying streptomycin, which. comprises treating a primary streptomycinsc'ontaining liquid with a water-soluble salt of a substantially water-insoluble carboxylic acid; recovering the precipitated salt-type combination of streptomycin andv thecarboxylic acid, and converting the latter combination into a water-soluble salt of streptomycin 10. The method. of purifying. streptomycin, which comprises treating a primary streptomycin-containing liquid with an activated charcoal, eluting the streptomycin from the charcoal with an aqueous, water-soluble mineral acid, treating the eluate with a water-soluble salt of a substantially water-insoluble carboxylic acid, recovering the precipitated salt-type combination of streptomycin and the carboxylic acidand converting the latter combination into a watersoluble salt of streptomycin.

11. The method which comprises mixing an aqueous solution of a water-soluble salt of streptomycin with an aqueous solution of a watersoluble salt of a substantially water-insoluble carboxylic acid.

12. The method of purifying streptomycin, which comprises treating an aqueous solution of an impure water-soluble salt of streptomycin with a water-soluble salt of a substantially water'- insoluble carboxylic acid, recovering the precipitated salt-type combination of streptomycin and the carboxylic acid, washing the latter combination with water, and converting it into a watersoluble salt of streptomycin.

13. The method which comprises treating an aqueous solution of a water-soluble salt of streptomycin having a pH of about 6.0 to about 8.0 with a water-soluble salt of a substantially waterinsoluble carboxylic acid.

14. The method which comprises treating an aqueous solution of a water-soluble salt of streptomycin having a pH around 7.0 with a watersoluble salt of a substantially water-insoluble carboxylic acid.

15. The method which comprises interacting a member of the group consisting of streptomycin and water-soluble salts thereof with a Watersoluble salt of a substantially water-insoluble fatty acid in'a solvent for the reactants.

16. The method of purifying streptomycin which comprises treating an impure member of the group consisting of streptomycin and watersoluble salts thereof with a water-soluble salt of a substantially water-insoluble fatty acid in a solvent for the reactants, recovering the precipitated salt-type combination of streptomycin and the fatty acid, and converting the latter combination into a water-soluble salt of streptomycin.

17. The method which comprises mixing an aqueous solution of a; water-soluble salt of streptomycin with an aqueous solution of a watersoluble salt of a substantially water-insoluble fatty acid.

18. The method of purifying streptomycin, which comprises treating an aqueous solution of an impure water-soluble salt of streptomycin with a water-soluble salt of a substantially waterinsoluble fatty acid, recovering the precipitated salt-type combination of streptomycin and the fatty acid, washing the latter combination with water, and converting it into a water-soluble salt of streptomycin;

WILLIAM BRAKER. WILLIAM A. LOTT.- ANDPEW ELLIS OKEEFFE.

REFERENCES CITED The following references are of record in the file-0f this patent:-

Fried et al.: Science, vol. 101 (1945'), pages 613-615, 3 pages.

Kuehl' et 'al.: Science, vol. 102 (1945), pages 34-35, 2 pages.

Kuehl et al.: JACS, vol. 68 (1946), pages 1460-1462. 

5. THE METHOD OF PURIFYING AN ANTIBIOTIC OF THE GROUP CONSISTING OF STREPTOMYCIN, DIHYDROSTREPTOMYCIN, STREPTOTHRICIN, AND WATER-SOLUBLE SALTS OF THESE WHICH COMPRISES TREATING AN AQUEOUS SOLUTION OF AN IMPURE ANTIBIOTIC OF THAT GROUP WITH A WATER-SOLUBLE SALT OF A SUBSTANTIALLY WATER-INSOLUBLE CARBOXYLIC ACID, RECOVERING THE PRECIPITATED SALT-TYPE COMBINATION OF THE ANTIBIOTIC AND THE CARBOXYLIC ACID, AND CONVERTING THE LATTER COMBINATION INTO A WATER-SOLUBLE SALT OF THE ANTIBIOTIC. 